The Rho GTPases Cdc42 and Rac signal to the actin cytoskeleton through proteins in the WASP family. Previous studies under the award discovered how the WASP autoinhibited domain is destabilized by Cdc42, leading to activation toward Arp2/3 complex, the cellular actin nucleation machine. Studies here will deepen our understanding of WASP regulation and address important new issues in biophysics, signal transduction and cell biology. Two unrelated WASP inhibitors have been discovered that appear to act by biasing the WASP autoinhibitory equilibrium to the inactive state. We will solve the structures of these molecules in complex with WASP, and learn at a quantitative level how they stabilize the autoinhibited domain, blocking Cdc42 and Arp2/3 complex interactions, through a series of biophysical assays. We will quantitate the effect of Cdc42 on phosphorylation and dephosphorylation kinetics of WASP, to learn if WASP could sense coincidence of GTPase and kinase/phosphatase signals. We will discover if phosphorylation enables WASP to be activated by SH2 domains in the Arp2/3 assay, and if SH2 domains act cooperatively with known WASP activators. We will use a battery of biophysical/ biochemical assays performed on a series of destabilized WASP proteins to correlate WASP stability, affinity for Cdc42 and activity toward Arp2/3, ultimately leading to a model for WASP regulation based on classical allostery. Finally, we will use NMR and biochemistry to discover how the WASP family member Scar, a target of Rac, is inhibited by the Pir121/NAP-1 complex, revealing common features of WASP family regulation. The work will lead to a structural and thermodynamic understanding of 2 mechanisms of signal integration by WASP, cooperative regulation by ligands (both protein and small molecule) that act on its autoinhibitory equilibrium, and contingent phosphorylation. It will reveal general structural and thermodynamic principles of autoinhibition, and how molecules regulated in this fashion may be used in the cell. The combined program will address fundamental issues in signal transduction, and could lead to new classes of cell biological probes and pharmaceutical agents that act through controlling conformational equilibria. Such molecules could be extremely valuable in studies of the cytoskeleton and treatment of diseases including metastatic cancer, immune system disorders and bacterial/viral infection.